The effect of phospholipase A2 inhibitors on proliferation and apoptosis of murine intestinal cells

Background. Group II phospholipase A(2) (PLA(2)) enzymes, the rate controlling enzymes in arachidonic acid metabolism, have been well characterized and subdivided into secretory 14-kDa PLA, (sPLA(2)) and cytoplasmic 85-kDa PLA(2) (cPLA(2)). Previous research has demonstrated increased PLA(2) in colorectal tumors. The present study was performed to determine the effect of specific PLA(2) inhibitors on the proliferation and induction of apoptosis of intestinal epithelial cells. Methods. A continuously proliferating rat small intestinal cell line (IEC-18) and a mouse colon cancer cell line (WB-2054-M-4) were utilized for these experiments. The cells were placed in microwells with serum-free or serum-supplemented media. The effects of serum on proliferation were then evaluated in the presence of the cPLA(2) inhibitor, methylarachidonyl fluorophosphate (MAFP), or the sPLA(2) inhibitor p-bromophenacyl bromide (BPB). The sPLA(2) and cPLA(2) protein was estimated by Western blotting. Proliferation of intestinal cells was quantitated by incorporation of [H-3]thymidine into DNA and PLA(2), activity was evaluated by quantitating arachidonic acid formation and prostaglandin E-2 production. Results. Western blotting of IEC-18 and WB-2054 cell protein demonstrated sPLA(2) and cPLA(2) enzyme in cells incubated in media containing 10% serum. Spontaneous DNA synthesis was present in both cell lines and serum consistently increased proliferation. In IEC-18 cells [H-3]thymidine incorporation stimulated by serum was inhibited by MAFP and BPB, while in the malignant cell line, proliferation was inhibited only by BPB. BPB, but not MAFP, produced a dose-dependent increase in apoptotic ratios in both cell lines. Arachidonic acid and PGE(2) formation, stimulated by serum, was inhibited by MAFP and BPB. Conclusions. A differential effect on intestinal cell mitogenesis was seen with different PLA(2) inhibitors. The sPLA(2), inhibitor, but not the cPLA(2) inhibitor, significantly inhibited [H-3]thymidine incorporation in the malignant cell line. This occurred with an induction of apoptosis. sPLA(2) inhibitors may be specific inhibitors of growth of malignant cells. The inhibition of arachidonic acid and PGE(2) production did not correlate with the inhibition of proliferation, suggesting that the two processes may be unrelated, (C) 1999 Academic Press.